lable at ScienceDirect

Soil Biology & Biochemistry 78 (2014) 284e287

Contents lists avai

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lbio

Short communication

Correction factors for dissolved organic carbon extracted from soil,measured using the Mn(III)-pyrophosphate colorimetric methodadapted for a microplate reader

Marc-Andr�e Giasson a, *, Colin Averill b, Adrien C. Finzi a

a Department of Biology, Boston University, Boston, MA 02215, USAb Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA

a r t i c l e i n f o

Article history:Received 30 April 2014Received in revised form6 August 2014Accepted 14 August 2014Available online 1 September 2014

Keywords:Colorimetric methodDOCForest soilsMn(III)-pyrophosphateOrganic carbon

* Corresponding author. Tel.: þ1 617 353 6993.E-mail addresses:mgiasson@bu.edu, marc.giasson.

http://dx.doi.org/10.1016/j.soilbio.2014.08.0110038-0717/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Oxidizable dissolved organic carbon (DOC) is regularly measured in environmental samples using acolorimetric method with Mn(III)-pyrophosphate as the oxidizing agent. It is simpler to use and has amuch higher throughput than the commonly used dichromate oxidation and combustion methods. Here,we demonstrate that the method often leads to an underestimation or overestimation of the concen-tration of common organic compounds in solutions. To our knowledge, no published study has taken thisfact into account when analyzing DOC data. Hence, we compared Mn(III)-pyrophosphate-based resultswith measurements performed with a total organic carbon combustion analyzer for samples of organicand mineral soil horizons of two temperate deciduous forests, of organic soil horizon of a primary-growth hemlock stand, and of a peatland located in New England, USA. The Mn(III)-pyrophosphatemethod consistently underestimated DOC concentration in soil extracts. We present correction factorsfor the different types of soil studied. By employing correction factors, we find the method can be aninexpensive, accurate, and high throughput tool to measure DOC in environmental samples.

© 2014 Elsevier Ltd. All rights reserved.

A colorimetric method developed by Bartlett and Ross (1988),using Mn(III)-pyrophosphate as the oxidizing agent, has beenwidely used to measure oxidizable dissolved organic carbon (DOC)in rain, stream, and soil water in ecosystems ranging from Arctictundra to peatlands, tropical forests, and a variety of agriculturalsystems (e.g., Xu et al., 2005; Gomes et al., 2012; Lipson et al., 2013;Martins Bezerra et al., 2013; Turner et al., 2013). Coupled withchloroform fumigation-extraction (Vance et al., 1987), it has alsobeen used extensively to estimate microbial biomass carbon in soils(e.g., da Silva et al., 2012; Gomes et al., 2012). Some organic com-pounds such as glycine and acetic acid resist oxidation (Bartlett andRoss, 1988), raising the concern that DOC concentration in envi-ronmental samples may be underestimated. In an extensive reviewof the literature, we searched the Web of Science database andGoogle Scholar on July 15, 2014, for all publications citing theoriginal methods paper by Bartlett and Ross (1988). We found 110journal articles and theses in which the Mn(III)-pyrophosphatemethod was used on environmental samples and found nonewhere authors mentioned any verification or correction for a

1@gmail.com (M.-A. Giasson).

possible underestimation of DOC concentration (SupplementaryTable 1). In this study, we adapted the Mn(III)-pyrophosphatecolorimetric method to use on a microplate reader and assessedits validity for soil extracts by comparing results to DOC measure-ments made with a total organic carbon (TOC) combustionanalyzer.

We collected soil samples from three locations in northeasternUnited States. Organic horizon (OH) and mineral soil (MS) sampleswere collected at the Hubbard Brook Experimental Forest, NewHampshire (43�560N, 71�450W) and Harvard Forest, Massachusetts(42�320N, 72�110W) in April, May, August, and October of 2012 and2013. At Hubbard Brook, samples were collected in a mature sugarmapleeyellow birch stand; soils were base-poor spodosols devel-oped on glacial till (Comerford et al., 2013). Harvard Forest sampleswere collected in a mature red oakered maple stand on a mixedmesic Typic Dystrochrept soil (Melillo et al., 2002). Samples were10� 10 cmOHmonoliths and 5 cm-diameter MS cores from the top15 cm of the mineral soil. At Harvard Forest, OH samples were alsocollected in a primary-growth hemlock stand in August 2013(Hadley and Schedlbauer, 2002).

Peat samples were removed from the Central Unit of CaribouBog, a 2200-ha peatland complex near Bangor, Maine (44�560N,68�460W). The Central Unit is an eccentric raised bog underlain

Table 1Regression coefficients of the linear relationships, shown on Fig. 1, between C con-centration of a few common organic compounds measured using the Mn(III)-pyrophosphate method and the expected C concentration.

Organiccompound

Slope Intercept r2 Concentrationrange applicable(mM C)

Acetic acid Not significantat P < 0.05

…

Citric acid 1.439 �0.008 0.997 0e3.2Fumaric acid 0.100 0.079 0.682 0e4Glucose 1.045 0.098 0.991 0e4Glutamine 0.300 �0.106 0.989 0e3.2Glycine 0.051 �0.008 0.431 0e4Malonic acid 1.335 �0.180 0.993 0e4Tannic acid 1.784 0.098 0.997 0e2.8

M.-A. Giasson et al. / Soil Biology & Biochemistry 78 (2014) 284e287 285

with glacio-marine clayesilt mineral soils (Comas et al., 2011). Wecollected 5 cm-diameter peat cores from the top 20 cm of the deadmoss layer in May, August, and October 2013 in sites dominated by(1) Sphagnum lawn, bryophytes, and sedges, (2) Sphagnum lawnand low ericaceous shrubs (Kalmia polifolia Wangenh., Rhododen-dron groenlandicum (Oeder) Kron& Judd) and (3) ericaceous shrubsand black spruce (Picea mariana (Mill.) B.S.P.). Soil characteristicsare presented in Supplementary Table 2.

Samples were immediately brought back to the laboratory andstored at 4 �C. OH and MS samples were homogenized by sievingthrough 2-mm mesh (4-mm mesh for hemlock OH) and removingrocks, roots, and woody debris. Roots were removed from peatsamples without sieving. Five grams each of OH and peat sample or10 g of MS sample were placed in 50-mL centrifuge tubes. 40 mL of0.5 M K2SO4 were added to each tube. Slurries were shaken for 1 hon an oscillating table and filtered through a Whatman #1 paperfilter. The extracts were frozen at �20 �C until DOC concentrationwas measured. Time between sample collection and freezing of theextracts was kept under 48 h.

To measure DOC, we adapted Bartlett and Ross's (1988) methodto use with a 96-well microplate reader. On each microplate, wepipetted duplicate 100-mL aliquots of eight oxalic acid standardswith concentrations ranging from 0- to 4-mM C and triplicate 100-mL aliquots of 24 soil extracts. We added 50 mL of 10 mM Mn(III)-pyrophosphate solution and 50 mL of concentrated sulfuric acid toeach well. The 96-well microplates were incubated in the dark atroom temperature for 18 h before measuring absorbance at 495 nmon a microplate reader (VersaMax, Molecular Devices, Sunnyvale,California, USA). For each microplate, soil extract absorbance wasconverted to C concentration using a linear calibration curve basedon the oxalic acid standards. We alsomeasured total organic carboncontent of the same soil extracts using an Apollo 9000 TOCAnalyzer with autosampler (Teledyne Tekmar, Mason, Ohio, USA).Arginine was used as a standard. Regression analyses were con-ducted inMatlab version 7.11.0 (MathWorks, Natick, Massachusetts,USA).

As a preliminary test, we assayed solutions of known concen-tration (0- to 4-mM C) of several organic compounds to determine

Fig. 1. Linear relationship between measured and expected C concentrations of eight comRegression coefficients and statistics are presented in Table 1. Only regressions statistically

if the colorimetric method measured all C present (Fig. 1, Table 1).Of the eight substances tested, only glucose and malonic acidshowed a stable recovery rate throughout the concentration range(Fig. 1a). The recovery rate of citric acid, glutamine, and tannic acidwas stable up to ~3-mM C after which the Mn(III)-pyrophosphatehad lost all its color and no further change in absorbance couldbe detected (Fig. 1b). In four of these five cases, the slope of thelinear regression between the measured and expected C concen-trations was greater than 1 (Table 1), indicating the method over-estimated the amount of C present. The recovery rate of glutaminewas less than 1; in this case C concentration was underestimated.Fumaric acid and glycine also had a recovery rate smaller than 1,and the linear regressions explained a much lower proportion ofthe variability (Table 1, Fig. 1c). Finally, acetic acid recovery rate wasnear zero and the linear regression was not statistically significant(Table 1, Fig. 1d). Given the high variability in the recovery rate ofthe various organic compounds and the great difficulty inmeasuring the relative abundance of these and other substances inenvironmental samples, it is highly probable that reported DOCconcentrations using the Bartlett and Ross (1988) colorimetricmethod are biased high or low depending on the particular mix ofDOC compounds present.

mon organic compounds estimated using a calibration curve based on oxalic acid.significant at P < 0.05 are shown.

Fig. 2. Linear relationship between DOC concentration of soil extracts measured usingthe Mn(III)-pyrophosphate method and a TOC combustion analyzer. The regressionsfor OH and peat samples, MS samples, and all samples aggregated are shown as lightgray, dark gray, and black solid lines, respectively. The dashed line represents the 1:1slope. Regression coefficients and statistics are presented in Table 2.

M.-A. Giasson et al. / Soil Biology & Biochemistry 78 (2014) 284e287286

To determine the magnitude of bias in soil samples collected inNew England ecosystems, we compared the DOC content of OH,MS, and peat samples measured using the colorimetric method anda TOC combustion analyzer (Fig. 2). Combustion methods areconsidered to yield absolute values of TOC and are often used as abenchmark to evaluate the efficiency of other methods (Nelson andSommers, 1996). Linear regressions between colorimetric andcombustion measurements explained most of the variation(r2 ¼ 0.62e0.87; Table 2). The colorimetric method underestimatedDOC concentration by 19e91% depending on the type and prove-nance of samples (Table 2). The underestimation differed betweensites and types of soil, presumably reflecting varying proportions ofthe different organic C compounds present, which in turn reflectdifferences in microbial populations and soil characteristics andprocesses.

A potassium dichromate colorimetric method is also widelyused to measure DOC in soil extracts (Nelson and Sommers, 1996).There is no need to apply a correction factor to that method ifsamples are heated for long enough because all C gets oxidized.

Table 2Regression coefficients of the linear relationships between DOC concentration of soilextracts measured using the Mn(III)-pyrophosphate method and a TOC combustionanalyzer for each site and type of soil. Mn(III)-pyrophosphate-based measurementscan be corrected using the equation DOCcorrected ¼ slope � DOCMn(III)-

pyrophosphate þ intercept. The correction is applicable for concentrations of0.08e4 mM C, the range over which the Mn(III)-pyrophosphate method gives validresults.

Site Forest type Soil type Slope Intercept r2

Hubbard Brook Sugar mapleeyellowbirch

Organic 1.393 �0.151 0.78

Hubbard Brook Sugar mapleeyellowbirch

Mineral 1.906 �0.938 0.77

Harvard Forest Red oakered maple Organic 1.190 0.428 0.62Harvard Forest Red oakered maple Mineral 1.481 0.153 0.79Harvard Forest Hemlock Organic 1.793 0.027 0.81Caribou Bog Raised bog Peat 1.449 0.135 0.87

All organicand peat

1.336 0.338 0.73

All mineral 1.715 �0.463 0.77All samples 1.447 0.140 0.75

This technique is more complicated to use, requires somespecialized equipment and more reagents, and has substantiallylower throughput than the Mn(III)-pyrophosphate method. DOCanalysis by combustion yields the most accurate measurements oforganic C (Nelson and Sommers, 1996), but it requires an expen-sive analyzer that also requires frequent maintenance andtroubleshooting.

Our results suggest that DOC in soil extracts can be successfullymeasured using the Mn(III)-pyrophosphate colorimetric method aslong as a correction for the bias is applied. When adapted to be runon an absorbance microplate reader, the Mn(III)-pyrophosphatecolorimetric method becomes an easy, inexpensive high-throughput technique allowing processing of several hundreds ofsamples per day. Because the correction factor varies depending onthe forest and type of soil studied, we recommend that a subset ofthe samples also be run on a combustion analyzer to determine thecorrection factor applicable. If this is impossible, at a minimum ageneral correction factor, such as the ones we present for NewEngland soils, should be used. We suggest the Mn(III)-pyrophosphate colorimetric method could also be used on othermatrices containing unknown amounts of C-containing com-pounds, such as rain and stream water, as long as a specificcorrection factor is estimated.

Acknowledgments

This researchwas supported by the National Science Foundation(131706-5056666) and by the Office of Science (BER), US Depart-ment of Energy, Terrestrial Ecosystem Science Program(SC0006916). We thank Rose Abramoff, Patrick Sorensen, andAllison Gill for their comments and suggestions during the prepa-ration of the manuscript. We are also grateful for their and severallab assistants' help with sample collection and processing.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.soilbio.2014.08.011.

References

Bartlett, R.J., Ross, D.S., 1988. Colorimetric determination of oxidizable carbon inacid soil solutions. Soil Science Society of America Journal 52, 1191e1192.

Comas, X., Slater, L., Reeve, A.S., 2011. Pool patterning in a northern peatland:geophysical evidence for the role of postglacial landforms. Journal of Hydrology399, 173e184.

Comerford, D.P., Schaberg, P.G., Templer, P.H., Socci, A.M., Campbell, J.L., Wallin, K.F.,2013. Influence of experimental snow removal on root and canopy physiologyof sugar maple trees in a northern hardwood forest. Oecologia 171, 261e269.

da Silva, M.S.C., da Silva, E.M.R., Pereira, M.G., da Silva, C.F., 2012. Litter stock and soilmicrobial activity in agroforestry systems. Floresta e Ambiente 19, 431e441 (inPortuguese).

Gomes, J.B.V., Fernandes, M.F., Barreto, A.C., de Araújo Filho, J.C., Curi, N., 2012. Soilattributes under agroecosystems and forest vegetation in the coastal tablelandsof northeastern Brazil. Ciencia e Agrotecnologia 36, 649e664.

Hadley, J.L., Schedlbauer, J.L., 2002. Carbon exchange of an old-growth easternhemlock (Tsuga canadensis) forest in central New England. Tree Physiology 22,1079e1092.

Lipson, D.A., Raab, T.K., Goria, D., Zlamal, J., 2013. The contribution of Fe(III) andhumic acid reduction to ecosystem respiration in drained thaw lake basins ofthe Arctic Coastal Plain. Global Biogeochemical Cycles 27, 399e409.

Martins Bezerra, R.P., Loss, A., Pereira, M.G., Perin, A., 2013. Forms of carbon in anoxisol under no-tillage and crop-livestock integration systems in the cerrado,Goias State, Brazil. Ciencias Agr�arias 34, 2637e2654 (in Portuguese).

Melillo, J.M., Steudler, P.A., Aber, J.D., Newkirk, K., Lux, H., Bowles, F.P., Catricala, C.,Magill, A., Ahrens, T., Morrisseau, S., 2002. Soil warming and carbon-cyclefeedbacks to the climate system. Science 298, 2173e2176.

Nelson, D.W., Sommers, L.E., 1996. Total carbon, organic carbon, and organic matter.In: Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N.,Tabatabai, M.A., Johnston, C.T., Sumner, M.E. (Eds.), Methods of Soil Analysis.

M.-A. Giasson et al. / Soil Biology & Biochemistry 78 (2014) 284e287 287

Part 3. Chemical Methods. Soil Science Society of America, Madison, WI,pp. 961e1010.

Turner, E.K., Worrall, F., Burt, T.P., 2013. The effect of drain blocking on the dissolvedorganic carbon (DOC) budget of an upland peat catchment in the UK. Journal ofHydrology 479, 169e179.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuringsoil microbial biomass C. Soil Biology and Biochemistry 19, 703e707.

Xu, X., Wang, Q., Hirata, E., 2005. Precipitation partitioning and related nutrientfluxes in a subtropical forest in Okinawa, Japan. Annals of Forest Science 62,245e252.